Cytoplasmic serine/threonine-specific kinases such as raf, protein kinase C, and mitogen activated protein (MAP) kinases play an integrative role in the transduction of signals from the cell membrane to the nucleus. To dissect raf-specific signaling pathways, we designed Raf-1 inhibition experiments employing monoclonal antibodies as well as expression of antisense RNA. The isolation of cells resistant to transformation by v- raf constituted a second strategy to identify factors that regulate raf- dependent signaling pathways. We conclude that Raf kinases function downstream of Ras, and that Raf-1 signaling is essential for transformation of NIH/3T3 cells by peripheral oncogenes and for regulation of a subset of early response genes by 12-0-tetradecanoylphorbol-13- acetate and serum growth factors. We have now determined the relative position of Raf and another Ras-controlled protein kinase family, the MAP or extracellular signal regulated kinase (ERK) kinases. c-Raf-1 and MAP kinases are serine/threonine protein kinases activated by numerous mitogens and extracellular agonists. The MAP kinases are regulated by phosphorylation at tyrosine and threonine, and are catalyzed by a novel kinase provisionally named MAP kinase-kinase (MAP-KK) c-Raf-1 is also regulated by phosphorylation and upon activation often complexes with receptor tyrosine kinases. We found that NIH/3T3 cells, stably transformed with v-raf, display constitutively active MAP kinases (42 and 44 kilodaltons) and MAP-KK. Moreover, c-Raf-1, immunoprecipitated from mitogen-treated cells or purified after baculoviral expression, can directly reactivate phosphatase-2A- inactivated MAP-KK in vitro. These results indicate that c-Raf-1 is the immediate proximal activator of MAP- KK.